"In vitro and in vivo estrogenicity of UV screens": response. (Correspondence).

We thank EHP for the opportunity to respond to the comments of Bolt and coworkers. Because Bolt et al. question our data on methodologic grounds and their use for risk assessment, we will deal with these two aspects separately.

Bolt et al. state that

in vitro data should serve only for screening purposes and that they are not suited for conclusions regarding risk assessment.

This should be clear from our paper. In vitro experiments certainly are not meant to serve for risk assessment; however, it is generally accepted that positive in vitro findings should lead to additional in vivo studies.

The oral experiments were conducted between spring and fall 1999. In contrast to Bolt et al.'s statement, there is still not an official guideline for the uterotrophic assay. The Endocrine Disrupters Testing and Assessment (EDTA) Task Force of the Organisation for Economic Co-operation and Development (OECD) decided on a proposal for the preparation of a guideline at its meeting held 26-27 May 2001.

Bolt et al. believe that our use of Long-Evans rats was not acceptable for our study. To our knowledge, the OECD protocol (1) will not specify the rat strains to be used for the uterotrophic assay. Long-Evans rats have long been used in neuroendocrine and endocrine investigations. The hairless (hr/hr) strain was recommended to us for dermatologic studies by a European breeding institute. Because we used this strain, we were able to use Aghazarian et al.'s (2) recent rat skin penetration data, which were obtained on skin of the same strain, for a provisional estimation of dermal dosage. The hr/hr rats are derived from the OFA (Oncins France)-SD (Sprague Dawley) strain (IFFA CREDO, L'Arbresle, France).

We used dermal application by immersion in warm olive oil after discussing the method with other endocrine disruptor experts. We chose this method mainly for the following reason: The surface on the back of the immature animal available for application of compound is much smaller than in adult rats, and plasters that would not unduly disturb the immature animals could easily be removed by the littermates in the cage. When working with immature animals, a major requirement is avoidance of stress. The OECD protocol (1) also recommends group housing "because single housing of immature animals may cause considerable stress." The procedure of immersion, carried out very gently, was well tolerated by the animals.

Irrespective of the method used to calculate the dermally applied dose, the amount taken up by the animal can never be calculated with the same precision as with the oral or parenteral routes from the amount applied. The only solution is the determination of blood and tissue levels, which presently is in progress in our laboratory using gas chromatography-mass spectrometry.

As we stated in our paper (3), experiments were conducted on offspring of time-pregnant rats. We recorded birth [occurring in the morning of gestational day (GD) 23, which is also postnatal day (PN) 1; GD 1 = 24 hr after mating period]; pups were then culled to eight per litter and observed daily. The development of uterine weight was studied in detail between PN20 and PN32 in Long-Evans and hairless rats (4). These data clearly show that in both strains, uterine weight remains at the same level until PN 26. Thus, PNs 25 and 26 can be used in our strains. This is also evident from an analysis of histograms of frequency distributions of individual uterine weights in controls and treated groups from oral and dermal studies investigated at PNs 25 and 26 (5).

With respect to OMC and Bp-3, our data are in full agreement with the data quoted by Bolt et al. The oral no-hormonal-effect levels Bolt et al. compared to our data were as follows: 250 vs. 522 mg/kg/day for OMC and 1,000 vs. 937 mg/kg/day for Bp-3. The lowest-observable-effect levels were 103 mg/kg/day for OMC and 1,525 mg/kg/day for Bp-3 in our oral study. The only difference is with 4-MBC, where no effect was seen after treatment with up to 1,000 mg/kg/day in the study quoted by Bolt et al., whereas we observed a significant increase in uterine weight at 119 mg/kg/day. Apart from rat strain, the main difference between the two studies is the route of application, subcutaneous versus oral (2). Even though the subcutaneous route is thought to be slightly more sensitive in the uterotrophic assay, this may not be the case for all compounds. Controversial uterine weight data also exist for other chemicals with proven binding and transcriptional activity at estrogen receptors. This issue cannot yet be considered to be completely settled. In some cases with negative uterine weight data, other estrogen-sensitive parameters were influenced by the compound (6). Recently, we corroborated increased uterine cell proliferation following dermal application of 4-MBC and OMC by demonstrating increased bromodeoxyuridine uptake (4,5).

In our view, Bolt et al. make inappropriate use of our data. There is international agreement that the uterotrophic assay can only serve a limited function as a test for in vivo identification of chemicals with estrogenic (or antiestrogenic) activity. To our knowledge, the uterotrophic assay is situated between in vitro screening tests and long-term studies (e.g., TG416) in the scenario for the investigation of endocrine disruptors conceived by the EDTA committee of the OECD, which initiated the validation of this test. The uterotrophic assay typically is an acute high-dose test. As discussed in detail in our paper (3), other known xeno-estrogens such as methoxychlor, nonylphenol, bisphenol A, and o,p'-DDT also need to be applied at similar high doses to achieve a significant growth of the uterus in a few days (7-10). If one considers the complex organizational and activational actions of steroid hormones at different stages of the life cycle, it becomes clear that such acute data cannot provide a basis for long-term risk calculations. In view of possible differences in gene regulation patterns, it is also not possible to draw conclusions on long-term risk from a comparison of dosages of different estrogenic chemicals.

At first sight, it seems tempting to relate estrogenic activities of different chemicals to a phytoestrogen occuring in food and to use this for a unified evaluation of "in vivo estrogenicity." However, this concept presents serious scientific flaws, in particular with regard to an application to long-term effects. It is evident from the scientific literature of the last 10-15 years that estrogenic chemicals are not alike. First of all, there are important differences in toxicokinetics that are not considered by comparing daily intake. In addition, and this may be more important, estrogenic chemicals can differ markedly in their effects on gene regulation, not only quantitatively but also qualitatively. It is well known that even closely related chemicals, such as tamoxifen and raloxifene, exhibit different tissue selectivity, acting as agonist (tamoxifen) or antagonist (raloxifene) in one and the same tissue (uterus) (11). Moreover, different compounds with agonistic or partial agonistic activity at estrogen receptors can recruit different coactivators/corepressors (11,12), and elicit different gene induction patterns, for example, in the uterus (13). In a recent study on adult ovariectomized rats (14), the effect of daidzein on mRNA expression in uterus differed qualitatively (up- or down-regulation) from that of bisphenol A for three out of six genes studied, and for DDT for two out of six genes. These differences in effects on gene regulation are not consistently revealed by simple measures of estrogenicity such as proliferation of cell lines or uterine growth. They may well matter in particular with long-term exposure. As a consequence, the principle of using a phytoestrogen such as daidzein (or ethinylestradiol) as a reference compound for estrogenic activity and extrapolating from the activity ratio calculated from acute data to long-term risk of chemicals with a different structure cannot be considered to represent a valid approach.

In our paper (3), we reported on acute in vitro and in vivo estrogen-like effects of UV screens. We deliberately abstained from extrapolation of these acute data to long-term exposure in view of risk assessment because this could not be considered to be scientifically sound on the basis of present knowledge. Much to our surprise, this is being done now by colleagues from both academia and industry. In our view, the calculation of safety margins for chronic exposure from our acute data is not acceptable on scientific grounds.

We insist that the effect levels of five UV screens in vitro and three compounds in the uterotrophic assay are in the range of other chemicals for which there is general agreement on the need for long-term studies on possible endocrine effects. Hence, we think that a solid risk assessment requires additional long-term studies, with particular reference to endocrine parameters, and a more detailed analysis of acute and chronic toxicokinetics.

REFERENCES AND NOTES

(1.) Protocol for the Conduct of the OECD Rodent Uterotrophic Assay. Unpublished data.

(2.) Aghazarian V, Tchiakpe L, Reynier JP, Gayte-Sorbier A. Release of benzimidazole and benzylidene camphor from topical sunscreen formulations. Drug Dev Ind Pharmacy 25:1277-1282 (1999).

(3.) Schlumpf M, Cotton B, Conscience M, Hailer V, Steinmann B, Lichtensteiger W. In vitro and in vivo estrogenicity of UV screens. Environ Health Perspect 109:239-244 (2001).

(4.) Schlumpf M, Berger L, Cotton B, Conscience-Egli M, Durrer S, Fleischmann I, Haller V, Markel K, Lichtensteiger W. Estrogen active UV screens. Seifen-Ole-Fette-Wachse (in press).

(5.) Berger L. Prepubertal Development of the Female Rat: Uterotrophic Response to Dermal Application of the UV Screens MBC, OMC, BP-3 and HMS [Diploma Thesis]. Zurich: University of Zurich, Faculty of Mathematical and Natural Sciences, 2001.

(6.) Gould JC, Leonard LS, Maness SC, Wagner BL, Conner K, Zacharewski T, Safe S, McDonnell DP, Gaido KW. Bisphenol A interacts with the estrogen receptor [alpha] in a distinct manner from estradiol. Mol Cell Endocrinol 142:203-214 (1998).

(7.) Metcalf JL, Laws SC, Cummings AM. Methoxychlor mimics the action of 17 [beta]-estradiol on induction of uterine epidermal growth factor receptors in immature female rats. Reprod Toxicol 10:393-399 (1996).

(8.) Shelby MD, Newbold RR, Tully DB, Chae K, Davis VL. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect 104:1296-1300 (1996).

(9.) Odum J, Lefevre PA, Tittensor S, Paton D, Routledge EJ, Beresford NA, Sumpter JP, Ashby J. The rodent uterotrophic assay: critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay. Regul Toxicol Pharmacol 25:176-188 (1997).

(10.) Ashby J, Tinwell H. Uterotrophic activity of bisphenol A in the immature rat. Environ Health Perspect 106:719-720 (1998).

(11.) Dutertre M, Smith CL. Molecular mechanisms of selective estrogen receptor modulator (SERM) action. J Pharmacol Exp The. 295:431-437(2000).

(12.) Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz KB. The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693-705(1997).

(13.) Nephew KP, Polek TC, Akcali KC, Khan SA. The antiestrogen tamoxifen induces c-fos and jun-B, but not c-jun or jun-D, protooncogens in the rat uterus. Endocrinology. 133:419-422 (1993).

(14.) Diel P, Schulz T, Smolnikar K, Strunck E, Vollmer G, Michna H. Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenic profiles and uterotropic activity. J Steroid Biochem Mol Biol 73:1-10 (2000).

Margret Schlumpf
Walter Lichtensteiger
Institute of Pharmacology and Toxicology
University of Zurich
Zurich, Switzerland
E-mail: [e-mail]